CD8؉ T-cells are a major source for the production of non-cytolytic factors that inhibit HIV-1 replication. In order to characterize further these factors, we analyzed gene expression profiles of activated CD8؉ T-cells using a human cDNA expression array containing 588 human cDNAs. mRNA for the chemokine I-309 (CCL1), the cytokines granulocyte-macrophage colony-stimulating factor and interleukin-13, and natural killer cell enhancing factors (

The serine protease inhibitors (serpins) are anti-inflammatory proteins that have various functions. By screening a diverse panel of viruses, we demonstrate that the serpin antithrombin III (ATIII) has a broad-spectrum anti-viral activity for HIV-1, HCV and HSV. To investigate the mechanism of action in more detail we investigated the HIV-1 inhibition. Using gene-expression arrays we found that multiple host cell signal transduction pathways were activated by ATIII in HIV-1 infected cells but not in uninfected controls. Moreover, the signal pathways initiated by ATIII treatment, were more than 200-fold increased by the use of heparin-activated ATIII. The most up-regulated transcript in HIV-1 infected cells was prostaglandin synthetase-2 (PTGS2). Furthermore, we found that over-expression of PTGS2 reduced levels of HIV-1 replication in human PBMC. These findings suggest a central role for serpins in the host innate anti-viral response. Host factors such as PTGS2 elicited by ATIII treatment could be exploited in the development of novel anti-viral interventions.

While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.While a new generation of vaccine vectors has been created to induce cellular immune responses, we know little about how to use them to maximize the generation of memory T-lymphocyte populations. Recombinant viral vectors and plasmid DNA constructs have been shown to elicit potent cellular immune responses (2,7,8,13,15,16,21,23). However, little has been done to evaluate the optimal routes of administration of these vaccine modalities and the relationship between the route of administration and in vivo vaccine antigen expression. Moreover, we do not fully understand the ramifications of the kinetics of vaccine antigen expression for immunogenicity.The in vivo evaluation of new vaccine vectors to select optimal routes of administration, dose, and biodistribution has been difficult, requiring serial sacrifice of laboratory animals and assessment of individual organs for vaccine antigen expression. To simplify this process of vaccine evaluation, we have adopted an in vivo imaging system (IVIS) to measure the expression of luciferase by vaccine vectors. This imaging strategy harnesses the ability of the luciferase protein to catalyze the light-producing oxidation of the small molecule luciferin. Luciferin is inoculated into mice that have received luciferaseexpressing immunogens, and the quantity of light emitted by this reaction is monitored in living mice (6).The use of in vivo imaging and luciferase expression (4), a technology developed for evaluating gene therapy strategies, is well suited for studying these novel vaccine vectors. We have recently shown that IVIS can be used to monitor the distribution and kinetics of vaccine vectors in the living mouse (12). The present study was done to explore the impact of the route of vaccine administration on vaccine antigen expression. In the process of doing these studies, we observed a striking association between the kinetics of antigen expression in vivo and the induction of long-term memory T cells.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of ␤-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8؉ cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8؉ T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8؉ T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1␣, MIP-1␤, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocytemacrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8؉ cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.

CD8 ؉ T-cells secrete soluble factor(s) capable of inhibiting both R5-and X4-tropic strains of human immunodeficiency virus type 1 (HIV-1). CCR5 chemokine ligands, released from activated CD8 ؉ T-cells, contribute to the antiviral activity of these cells. These CC-chemokines, however, do not account for all CD8 ؉ T-cell antiviral factor(s) (CAF) released from these cells, particularly because the elusive CAF can inhibit the replication of X4 HIV-1 strains that use CXCR4 and not CCR5 as a coreceptor. Here we demonstrate that activated CD8 ؉ T-cells of HIV-1-seropositive individuals modify serum bovine antithrombin III into an HIV-1 inhibitory factor capable of suppressing the replication of X4 HIV-1. These data indicate that antithrombin III may play a role in the progression of HIV-1 disease.Soluble inhibitory factors produced by CD8 ϩ T-cells have been shown to inhibit HIV-1 1 replication and may play a critical role in vivo in antiviral host defense (1). These inhibitory factors include CC-chemokines (2-4), which bind to the CCR5 coreceptor and inhibit R5 viral entry into cells (1) (5-7), as well as less well characterized soluble factor(s) produced by CD8 ϩ T-cells and termed CD8 ϩ T-cell antiviral factor(s) (CAF), which are capable of inhibiting both R5 and X4 .Recently, we demonstrated that there are two factors produced by activated CD8 ϩ T-cells capable of inhibiting the X4 strain HIV-1 IIIB (16). These factors are distinctive in size and the ability to bind heparin. One of these factors bound heparin at physiological salt concentration, eluted at 350 mM NaCl, and was retained by a 50-kDa cut-off Centricon filter. The other factor did not bind heparin at physiological salt concentration and was filtered through a 50-kDa cut-off Centricon filter. The HIV-1 inhibitory activity of these factors was higher with bulk CD8 ϩ T-cells of seropositive individuals and HIV-1-specific cytotoxic T-lymphocytes (CTL) compared with bulk CD8 ϩ Tcells of HIV-1-seronegative individuals (16). In the present study we identified the heparin binding inhibitory activity as a CD8 ϩ T-cell modified form of antithrombin, which is produced in higher amounts by HIV-1-specific CTL and bulk CD8 ϩ Tcells of seropositive individuals than by bulk CD8 ϩ T-cells of seronegative individuals. In this study for the first time we demonstrate that CD8 ϩ T-cells can activate a serum protein to become inhibitory for HIV, a possibility that has not been addressed previously. EXPERIMENTAL PROCEDURESHIV-specific CTL Clones and Bulk CD8 ϩ T-Cells-Polyclonal CD8 ϩ cells that were 90 -99% CD3 ϩ -and CD8 ϩ -positive were generated by fluorescence-activated cell sorting from the six seronegative and six HIV-1-seropositive long-term nonprogressors by positive selection with anti-CD8 antibody-coated immunomagnetic beads (PerSeptive Biosystems, Framingham, MA) as described (16). HIV-1-specific CTL clones were used as described (16). Bulk CD8 ϩ cell lines from seropositive and seronegative persons were established by incubating purified CD8 ϩ cells (2 ϫ 10 6 )...

IntroductionPlasmid DNA vaccines are a promising modality for immunization against a variety of infectious agents because they are safe, readily scalable, and easy distributed. Plasmid DNA vaccine vectors can elicit CD8 ϩ cytotoxic T lymphocytes (CTLs), CD4 ϩ T helper cell immune responses, as well as humoral immune responses. Nonetheless, the utility of DNA immunogens has been limited by their failure to elicit sufficiently potent immune responses. 1 One potential explanation for the limited immunogenicity of plasmid DNA is that vaccine antigen expression is generated at only transient and at low levels. 1 Immune-mediated destruction of antigen-producing muscle fibers appears to play a significant role in limiting vaccine antigen expression. Clearance of antigen-expressing myocytes has been shown to be dependent both on the immunogenicity of the antigen and the presence of a functional immune system. 2,3 However, the cell types responsible for this destruction remain to be determined. We have shown that damping of plasmid DNA vaccine antigen expression in vivo occurs coincident with the emergence of major histocompatibility complex (MHC) class I-restricted T-cell responses. In addition, we observed that vaccine antigen expression persists in Fas receptor knockout mice, suggesting a role in this process for T cell-mediated apoptosis via the Fas/FasL pathway. 3 Based on these data, we hypothesized that CD8 ϩ T cells mediated vaccine antigen clearance through Fas-dependent apoptosis. Alternatively, other studies have suggested that the limited antigen expression in this setting may be a result of antibody-dependent cell-mediated cytotoxicity or complement-mediated lysis. 4 In addition to adaptive immune responses, innate immune responses, such as those mediated by macrophages and NK cells, have also been implicated as potential contributors to the destruction of antigenproducing myocytes. 5,6 In the present study, we investigated the cell types responsible for antigen clearance in plasmid DNA vaccinated mice. We used an In Vivo Imaging System (IVIS), which enabled us to measure antigen expression in vivo precisely, without serial killing of the animals. Using knockout (KO) mice and antibodydepletion experiments, we investigated the relative contribution of NK cells, macrophages, CD8 ϩ T cells, and CD4 ϩ T cells to the damping of antigen expression in vaccinated animals. Surprisingly, we observed that CD4 ϩ T cells were both necessary and sufficient to mediate plasmid DNA vaccine antigen clearance. These findings demonstrate a central role for CD4 ϩ T cells in vaccine antigen clearance. Methods Animals and immunizationsSix-to 8-week-old wild-type C57BL/6, C57BL/6.␤2 M KO, C57BL/ 6.MHC II KO, Rag1 KO, and NK-function-deficient beige mice (C57BL/6-Lyst bg7-9 ) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed and maintained in accordance with the Guide for the Care and Use of Laboratory Animals, 10 and all studies and procedures were reviewed and approved by the Institutional Animal...

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Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.